This invention provides general methods for discovering mutant inhibitors for any class of enzymes as well as the specific inhibitors so identified. More specifically, this invention provides general methods for discovering specific inhibitors for multi-substrate enzymes. Examples of such multi-substrate enzymes include, but are not limited to, kinases and transferases. The mutant inhibitors identified by the methods of this invention can be used to highly selectively disrupt cell functions such as oncogenic transformation. In one particular example, this invention provides a Src protein kinase inhibitor, pharmaceutical compositions thereof and methods of disrupting transformation in a cell that expresses the target v-Src comprising contacting the cell with the protein kinase inhibitor.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. U.S. patent application Ser. Nos. 08/797,522 and 60/046,727, and PCT/US98/02522 are related to the present invention and each of these applications is specifically and individually incorporated by reference in its entirety.
The current explosion in the number of newly discovered genes underscores the need for small molecule ligands which can be used to elucidate and control gene function. Convergent engineering of protein/small molecule interfaces has emerged in recent years as a powerful method for generating novel ligand/receptor pairs with high specificity. By introducing chemical diversity into the target protein as well as the small molecule, unique binding interactions can be designed and exploited more efficiently than through traditional medicinal chemistry. Such approaches have been used to chemically explore a number of biological systems. FK506-binding protein has been engineered to preferentially bind non-natural FK506 analogues by Schreiber and co-workers, as well as Clackson and co-workers. This system has been used extensively to selectively dimerize receptors and control gene expression in a cellular context. Nuclear hormone receptors have also been shown to be amendable to chemical genetic design. Corey and co-workers demonstrated that mutations at two amino acid residues in the retinoid X receptor are sufficient to create two new classes of receptors with novel ligand specificities. In a more medicinally applicable system, Smith and co-workers engineered the protease, carboxypeptidase A1, to hydrolyze a prodrug of methotrexate which is resistant to hydrolysis by wild type proteases.
Protein kinase catalyzed phosphorylation of the hydroxyl moiety of serine, threonine or tyrosine is the central post-translational control element in eukaryotic signal transduction. The phosphorylation state of a given protein can govern its enzyme activity, protein-protein binding interactions, and cellular distribution. Phosphorylation and dephosphorylation is thus a xe2x80x9cchemical switchxe2x80x9d which allows the cell to transmit signals from the plasma membrane to the nucleus to ultimately control gene expression in a highly regulated manner. Highly selective, cell permeable inhibitors of individual kinases would allow for the systematic investigation of the cellular function of a kinase in real time, and thus, would provide invaluable tools for the deconvolution of phosphorylation dependent processes in signal transduction cascades.
The Src family is composed of ten highly homologous cytosolic kinases which are critical components in an array of cell signaling pathways ranging from lymphocyte activation to cell growth and proliferation. Constitutive activation of these enzymes can lead to oncogenic cell transformation, making them putative drug targets for cancer therapies. Because of their importance in the regulation of these fundamental cellular processes, many studies have focused on developing inhibitors for the Src family kinase. However, the potent inhibitors that have been discovered lack the high selectivity that would be required for probing the cellular inhibition of an individual target kinase. Conventional inhibitor screens have produced few if any molecules which can discriminate between the active sites of the various Src family kinases.
Unfortunately, the very features which make kinases so useful in signal transduction, and which has made them evolve to become central to almost every cellular function, also makes them extremely difficult, if not impossible, to study and understand. Their overlapping protein specificities, their structural and catalytic similarities, their large number, and their great speed make the specific identification of their in vivo protein substrates extremely difficult, if not impossible, using current genetic and biochemical techniques. This is today the main obstacle to deciphering the signaling cascades involved in protein kinase-mediated signal transduction (4,6-8).
Efforts to dissect the involvement of specific protein kinases in signal transduction cascades have been frustrated by their apparent lack of protein substrate specificity in vitro and in vivo (4,8). The catalytic domains of protein kinases possess little or no inherent protein substrate specificity, as demonstrated by domain swapping experiments (18-23). The catalytic domain from one protein kinase can be substituted into a different protein kinase with little change in the protein substrate specificity of the latter (22).
The poor in vitro specificity of kinases also makes it difficult, if not impossible, to extrapolate what the in vivo function of given kinases might be. An isolated protein kinase of interest will often phosphorylate many test substrates with equal efficiency (29). This apparently poor substrate specificity is also found in vivo; for example, many genetic approaches, such as gene knock out experiments, give no interpretable phenotype due to compensation by other cellular protein kinases (30,31).
Another complication is that many protein kinases have been proposed to phosphorylate downstream and upstream proteins which are themselves protein kinases; although this appears to make complex positive feedback loops possible, it also makes dissecting the cascade even more difficult (1). One important avenue for deciphering the role and understanding the function of enzymes, both in vitro and in vivo, is the use of specific enzyme inhibitors. If one or more compound can be found that will uniquely inhibit the protein kinase target, the inhibitor can be used to modulate the enzyme""s activity, and the effects of that decrease can be observed. Whole genome techniques have provided many targets but their function is unknown. Many methods have been developed to determine if a given new kinase could be a good target. These methods, all have in common the lack of a small molecule to inhibit the enzyme which can lead to confusion.
For example, the most commonly used state of the art technique is to knock out the kinase and see a new phenotype. Typically, deletion of one kinase in the mouse genome (most common model organism) causes no informative change. This is for two reasons: 1) either the gene kinase) is essential during embryogenesis, thereby causing lethality before birth, or 2) the gene is absent (knocked out) and its function can be replaced by a closely related kinase which is still present. The important difference between the art recognized approach and the invention herein is that herein small organic molecules are employed to inhibit the function of the kinase of interest, since it is still present in the organisms but inactive thus it can cause significant changes to the organisms and most importantly the changes are exactly like that which would occur if an inhibitor of wild-type enzyme was made.
In addition, such inhibitors are among the most important pharmaceutical compounds known. For example, aspirin (acetylsalicylic acid) is such an inhibitor. It inhibits an enzyme that catalyzes the first step in prostaglandin synthesis, thus inhibiting the formation of prostaglandins, which are involved in producing pain (72). Traditional drug discovery can be characterized as the design and modification of compounds designed specifically to bind to and inactivate a disease-causing protein; the relative success of such an effort depends upon the selectivity of the drug for the target protein and its lack of inhibition of non-disease associated enzymes with similar enzyme activities. Such approaches would appear to be promising ways to develop treatments for cancer, since many human cancers are caused by disregulation of a normal protein (e.g., when a proto-oncogene is converted to an oncogene through a gene translocation). And since kinases are key regulators, they have turned out to be very common proto-oncogenes, and thus ideal drug design targets.
The process of designing selective inhibitors is relatively simple in cases where few similar enzymes are present in the target organism, for example in cases where inhibitors of a protein unique to bacteria can be targeted. But unfortunately, the similarities between the kinases and their large number has almost completely frustrated the discovery and design of specific inhibitors, and has blocked most hopes of developing specific pharmaceutical treatments aimed at the proto-oncogene level. It is expected that the vast majority of candidate inhibitors will inhibit multiple kinases, even though they may have initially been identified as inhibiting a particular, purified kinase.
These difficulties described above have implications well beyond the mere frustration of scientists; they have frustrated efforts to decipher the kinase cascades and the function of individual kinases in those cascades and other cellular mechanisms. Such an understanding of kinase activity and function may be essential before certain human diseases can be effectively treated, prevented or cured. For example, it has been known for over 30 years that the oncogene bcr-abl is a protein kinase that is responsible for chronic myelogenous leukemia; but the physiological substrates that it acts upon to cause oncogenesis, which may be important drug design targets, have yet to be definitively identified (11). On the bright side, despite this shortcoming, the inhibitor CGP 57148 is reportedly now undergoing clinical trials for use in treating myelogenous leukemia, even though the substrates it may block phosphorylation of in vivo are not known.
The medical significance of these difficulties is further illustrated by the Rous sarcoma virus (RSV), which has become an important model system for studying the role of kinases in oncogenesis. RSV transformation of fibroblasts is controlled by a single viral gene product, the protein kinase v-src (32). It is the rapid time course and the dramatic morphological changes during RSV fibroblast transformation that have made RSV a paradigm for studies of oncogene activity in all cells. The origin (33), regulation (3,8,34,35), and structure (25,27,36) of v-Src have been extensively studied and are well understood (8,37,38). But central questions about the intensely studied kinase remains unanswered: what are its direct cellular substrates? Does inhibition of its catalytic activity effectively inhibit, or even reverse, transformation? Would such inhibition be an effective therapy for or prophylactic against RSV transformation? Unfortunately, as discussed above, the answers to these questions are not forthcoming, largely because the number of cellular kinases is enormous (it is estimated that 2% of the mammalian genome encodes protein kinases (4)) and because protein kinases display overlapping substrate specificities (8,39) and share catalytic domains, making the design of specific inhibitors enormously difficult.
Although the difficulties are daunting, new methods of rational drug design and combinatorial organic synthesis make the design or discovery of kinase-specific inhibitors feasible given sufficient resources. However, because the kinase networks are highly degenerate and interconnected in unknown ways, there is considerable uncertainty with regard to many diseases which kinases should be targeted for inhibition. Moreover, it is by no means clear that a specific inhibitor of a given kinase will have any effect on the disease, either in vitro or in vivo. Because kinases can be highly promiscuous, there is a significant chance that inhibiting one kinase will simply force another kinase to xe2x80x9ctake its place.xe2x80x9d
The present invention provides a strategy (i.e., methodology) of combining chemical and genetic approaches to enable the rapid generation of highly selective small molecule inhibitors for one engineered enzymes, such as kinases and methyltransferases, in vitro and in whole cells. The invention disclosed herein involves using a specific point mutation to create a unique pocket in the substrate binding pocket or site of the enzyme of interest which does not occur in any other enzyme in the genome. A specific inhibitor of the engineered enzyme is then synthesized by derivatizing an enzyme inhibitor with a bulky group designed to fit the novel active site pocket. By using genetic manipulation to introduce a unique structural difference into the conserved enzyme active site, highly selective inhibitors can be identified from very small panels (10 compounds) of putative inhibitors as explained herein. The inhibitors of the present invention are useful for studying the function of enzymes in biochemical pathways as well as for therapeutic purposes.
This invention provides inhibitors that do not inhibit a catalytic activity of a wild-type enzyme but do inhibit the same catalytic activity of the corresponding mutant enzyme, wherein the wild-type enzyme and the mutant enzyme are functionally identical. More specifically, the inhibitors of the present invention inhibit the catalytic activity of a mutant enzyme with an IC50 of less than about 200 nM. The present invention further provides methods of inhibiting a catalytic activity of a mutant enzyme by contacting the mutant enzyme with the inhibitors of this invention.
The present invention provides inhibitors that do not inhibit the growth of a cell expressing a wild-type enzyme but do inhibit the growth of a cell expressing a mutant form of the wild-type enzyme, wherein the wild-type enzyme and the mutant form of the wild-type enzyme are functionally identical. Examples of inhibitors provided by the present invention include, but are not limited to, protein kinase inhibitors, lipid kinase inhibitors, aminoglycoside kinase inhibitors and transferase inhibitors, such as methyltransferase inhibitors. The present invention also provides methods of inhibiting the growth of a cell expressing a mutant enzyme by contacting a cell with the inhibitors of the present invention.
The present invention further provides protein kinase inhibitors represented by the following formula I: 
wherein R is a 1xe2x80x2-naphthyl; 2xe2x80x2-napthyl; m-phenoxyphenyl; m-benzyloxyphenyl; m-(2xe2x80x2, 6xe2x80x2-dichloro)benzyloxyphenyl; 3-piperonylpyrazolo; p-tert-butylphenyl; 1xe2x80x2-naphthylmethyl; 1xe2x80x2-napthoxymethyl; or 2xe2x80x2-naphthylmethyl. More specifically, the present invention provides such protein kinase inhibitors where R is 1xe2x80x2-naphthyl; 2xe2x80x2-naphthyl or 1xe2x80x2-napthylmethyl; 2xe2x80x2-napthylmethyl. The present invention also provides compositions which include the protein kinase inhibitors of the present invention.
The present invention provides methods of disrupting transformation in a cell that expresses a mutant protein kinase of the Src family by contacting the cell with the protein kinase inhibitors of the present invention. More specifically, the present invention provides methods of disrupting transformation in a cell that expresses I338G v-Src or T339G Fyn by contacting the cell with the protein kinase inhibitors of the present invention.
The present invention further provides methods of disrupting transformation in a cell that expresses a mutant protein kinase of the Src family by contacting the cell with a composition comprising the protein kinase inhibitors of the present invention. More specifically, the present invention provides methods of disrupting transformation in a cell that expresses I338G v-Src or T339G Fyn by contacting the cell with a composition comprising the protein kinase inhibitors of the present invention.
The present invention also provides methods of inhibiting the phosphorylation of a substrate of a mutant protein kinase by incubating a protein kinase inhibitor of the present invention with a mixture containing the mutant protein kinase and its substrate.
The present invention also provides methods of inhibiting the catalytic activity of a mutant enzyme by incubating the mutant enzyme with an inhibitor of the present invention.
The present invention also provides methods of inhibiting the growth of a cell by incubating the cell with an inhibitor of the present invention.
Mutant protein kinases used in the methods of the present invention include, but are not limited to the following: i) mutant protein kinases of the Src family, such as mutant v-Src; ii) mutant Fyn; iii) mutant c-Abl; iv) mutant CAMK IIxcex1; v) mutant CDK2; vi) mutant Cdc28 and vii) mutant Fus3. Specific examples of mutant protein kinases used in the methods of the present invention include, but are not limited to the following: i) I338G v-Src; ii) T339G Fyn; iii) T315A Abl; iv) F89G CAMK IIxcex1; v) F80G CDK2; vi) Cdc28-as1 and vii) Fus-as1.
This invention further provides a general approach for sensitizing protein kinases to cell permeable molecules which do not inhibit any wild-type protein kinases. Using this approach, potent and specific inhibitors from two structural classes of putative inhibitors are identified for seven protein kinases from five distinct sub-families. This approach can be used in vivo to systematically generate conditional alleles of protein kinases.
This invention also provides mutant kinase, Cdc28-as1 (analog-specific 1), that is uniquely sensitive to the cell-permeable inhibitor 4-amino-1-(tert-butyl)-3-(1xe2x80x2-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NM-PP1). In cdc28-as1 cells, entry into mitosis is inhibited by low concentrations of 1-NM-PP 1, whereas higher concentrations of inhibitor are required to induce the G1 arrest that is typically observed in temperature-sensitive cdc28 mutants. Genome-wide transcriptional analysis confirms that 1-NM-PP1 treatment of cdc28-as1 cells leads to inhibition of G2/M-specific gene expression, whereas treatment of wild-type cells has no significant effects. The generation of the analog-specific cdc28-as1 mutant thus provides a highly specific method for inhibiting Cdc28 activity in the cell, and demonstrates the general utility of this method in the analysis of protein kinases.